[Comment-Thomas: note, to-dos are now mostly listed in margin notes on right.]

1 Introduction

1.1 Motivation

The spatialHeatmap package provides functionalities for visualizing cell-, tissue- and organ-specific data of biological assays by coloring the corresponding spatial features defined in anatomical images according to a numeric color key. The color scheme used to represent the assay values can be customized by the user. This core functionality of the package is called a spatial heatmap (SHM) plot. It is enhanced with nearest neighbor visualization tools for groups of measured items (e.g. gene modules) sharing related abundance profiles, including matrix heatmaps combined with hierarchical clustering dendrograms and network representations. The functionalities of spatialHeatmap can be used either in a command-driven mode from within R or a graphical user interface (GUI) provided by a Shiny App that is also part of this package. While the R-based mode provides flexibility to customize and automate analysis routines, the Shiny App includes a variety of convenience features that will appeal to experimentalists and other users less familiar with R. Moreover, the Shiny App can be used on both local computers as well as centralized server-based deployments (e.g. cloud-based or custom servers) that can be accessed remotely as a public web service for using spatialHeatmap’s functionalities with community and/or private data. The functionalities of the spatialHeatmap package are illustrated in Figure 1.

Functionality overview of spatialHeatmap. This figure illustrates the overview of spatialHeatmap functionality on a gene expression data, which could be generalized on many other data types. The accepted data classes are `SummarizedExperiment` (SE, A1), `data frame` (A2), `vector` (A3). If exist, replicates should be aggregated. Subfigure B is the aSVG image (see [aSVG](#term) below), where target spatial features (*e.g.* S1) are pre-annotated. The numeric values of target samples in data are mapped to matching features in aSVG and translated to colors (*e.g.* S1), and the resulting plots are spatial heatmaps (SHMs, subfigure C). The color scale on the left indicates gene expression levels and the legend plot on the right labels target spatial features. As a supplement to SHMs, the target gene is explored in the context of its coexpression cluster in form of matrix heatmap (subfigure D) and network (subfigure E). In addition to command-driven mode, all utilities are also combined as an interactive Shiny App, which would be convenient for users with litte programing exprience.

Figure 1: Functionality overview of spatialHeatmap
This figure illustrates the overview of spatialHeatmap functionality on a gene expression data, which could be generalized on many other data types. The accepted data classes are SummarizedExperiment (SE, A1), data frame (A2), vector (A3). If exist, replicates should be aggregated. Subfigure B is the aSVG image (see aSVG below), where target spatial features (e.g. S1) are pre-annotated. The numeric values of target samples in data are mapped to matching features in aSVG and translated to colors (e.g. S1), and the resulting plots are spatial heatmaps (SHMs, subfigure C). The color scale on the left indicates gene expression levels and the legend plot on the right labels target spatial features. As a supplement to SHMs, the target gene is explored in the context of its coexpression cluster in form of matrix heatmap (subfigure D) and network (subfigure E). In addition to command-driven mode, all utilities are also combined as an interactive Shiny App, which would be convenient for users with litte programing exprience.

jianhai-reply: legend is edited.1 Thomas-Comment: the legend text of Figure 1 should provide a brief overview of what is shown in the flowchart. Right now there is too much technical detail that lacks context. I suggest to significantly shorten it. Also, you may want to organize the flowchart with bullet labels like (A), (B), (C) and (D) that can be referenced in the text. For instance: (A) Expression Data, (B) aSVG, (C) SHM and (D) Gene Context Plots. This way there is a clear label plus a visual illustration for each component one can refer to in the text with Figure 1A or Figure 1B, etc.

As anatomical images the package supports both tissue maps from public repositories and custom images provided by the user. In general any type of image can be used as long as it can be provided in SVG (Scalable Vector Graphics) format, where the corresponding spatial features have been defined (see aSVG below). The numeric values plotted onto an SHM are usually quantitative measurements from a wide range of profiling technologies, such as microarrays, next generation sequencing (e.g. RNA-Seq and scRNA-Seq), proteomics, metabolomics, or many other small- or large-scale experiments. For convenience, several preprocessing and normalization methods for the most common use cases are included that support raw and/or preprocessed data. Currently, the main application domains of the spatialHeatmap package are numeric data sets and spatially mapped images from biological, agricultural and biomedical areas. Moreover, the package has been designed to also work with many other spatial data types, such a population data plotted onto geographic maps. This high level of flexibility is one of the unique features of spatialHeatmap. Related software tools for biological applications in this field are largely based on pure web applications (Winter et al. 2007; Waese et al. 2017) or local tools (Maag 2018; Muschelli, Sweeney, and Crainiceanu 2014) that typically lack customization functionalities. These restrictions limit users to utilizing pre-existing expression data and/or fixed sets of anatomical image collections. To close this gap for biological use cases, we have developed spatialHeatmap as a generic R/Bioconductor package for plotting quantitative values onto any type of spatially mapped images in a programmable environment and/or in an intuitive to use GUI application.

1.2 Design

The core feature of spatialHeatmap is to map the assay values (e.g. gene expression data) of one or many items (e.g. genes) measured under different conditions in form of numerically graded colors onto the corresponding cell types or tissues represented in a chosen SVG image. In the gene profiling field, this feature supports comparisons of the expression values among multiple genes by plotting their SHMs next to each other. Similarly, one can display the expression values of a single or multiple genes across multiple conditions in the same plot (Figure 3). This level of flexibility is very efficient for visualizing complicated expression patterns across genes, cell types and conditions. In case of more complex anatomical images composed of overlapping multiple layer tissues, it is important to visually expose the tissue layer of interest in the plots. To address this, several default and customizable layer viewing options are provided. They allow to hide features in the top layers by making them transparent in order to expose features below them. This transparency viewing feature is highlighted below in the mouse example (Figure 6).

To maximize reusability and extensibility, the package organizes large-scale omics assay data along with the associated experimental design information in a SummarizedExperiment object. The latter is one of the core S4 classes within the Bioconductor ecosystem that has been widely adapted by many other software packages dealing with gene-, protein- and metabolite-level profiling data (Morgan et al. 2018). In case of gene expression data, the assays slot of the SummarizedExperiment container is populated with a gene expression matrix, where the rows and columns represent the genes and tissue/conditions, respectively, while the colData slot contains metadata including replicate information. The tissues and/or cell type information in the object maps via colData to the corresponding features in the SVG images using unique identifiers for the spatial features (e.g. tissues or cell types). This allows to color the features of interest in an SVG image according to the numeric data stored in a SummarizedExperiment object. For simplicity the numeric data can also be provided as numeric vectors or data.frames. This can be useful for testing purposes and/or the usage of simple data sets that may not require the more advanced features of the SummarizedExperiment class, such as measurements with only one or a few data points. Details about how to access the SVG images and properly format the associated expression data are provided in the Supplement section of this vignette.

1.3 Image Format: SVG

SHMs are images where colors encode numeric values in features of any shape. For plotting SHMs, Scalable Vector Graphics (SVG) has been chosen as image format since it is a flexible and widely adapted vector graphics format that provides many advantages for computationally embedding numerical and other information in images. SVG is based on XML formatted text describing all components present in images, including lines, shapes and colors. In case of biological images suitable for SHMs, the shapes often represent anatomical or cell structures. To assign colors to specific features in SHMs, annotated SVG (aSVG) files are used where the shapes of interest are labeled according to certain conventions so that they can be addressed and colored programmatically. SVGs and aSVGs of anatomical structures can be downloaded from many sources including the repositories described below. Alternatively, users can generate them themselves with vector graphics software such as Inkscape. Typically, in aSVGs one or more shapes of a feature of interest, such as the cell shapes of an organ, are grouped together by a common feature identifier. Via these group identifiers one or many feature types can be colored simultaneously in an aSVG according to biological experiments assaying the corresponding feature types with the required spatial resolution. Correct assignment of image features and assay results is assured by using for both the same feature identifiers. The color gradient used to visually represent the numeric assay values is controlled by a color gradient parameter. To visually interpret the meaning of the colors, the corresponding color key is included in the SHM plots. Additional details for properly formatting and annotating both aSVG images and assay data are provided in the Supplement section of this vignette.

1.4 Data Repositories

If not generated by the user, SHMs can be generated with data downloaded from various public repositories. This includes gene, protein and metabolic profiling data from databases, such as GEO, BAR and EBI. A particularly useful resource, when working with spatialHeatmap, is the Expression Atlas from EMBL-EBI (Papatheodorou et al. 2018). This online service contains both assay and anatomical images. Its assay data include mRNA and protein profiling experiments for different species, tissues and conditions. The corresponding anatomical image collections are also provided for a wide range of species including animals and plants. In spatialHeatmap several import functions are provided to work with the expression and aSVG repository from the Expression Atlas directly. The aSVG images developed by the spatialHeatmap project are available in its own repository called spatialHeatmap aSVG Repository, where users can contribute their aSVG images that are formatted according to our guidlines.

1.5 Tutorial Overview

The following sections of this vignette showcase the most important functionalities of the spatialHeatmap package using as initial example a simple to understand toy data set, and then more complex mRNA profiling data from the Expression Atlas and GEO databases. First, SHM plots are generated for both the toy and mRNA expression data. The latter include gene expression data sets from RNA-Seq and microarray experiments of Human Brain, Mouse Organs, Chicken Organs, and Arabidopsis Shoots. The first three are RNA-Seq data from the Expression Atlas, while the last one is a microarray data set from GEO. Second, gene context analysis tools are introduced, which facilitate the visualization of gene modules sharing similar expression patterns. This includes the visualization of hierarchical clustering results with traditional matrix heatmaps (Matrix Heatmap) as well co-expression network plots (Network). Third, an overview of the corresponding Shiny App is presented that provides access to the same functionalities as the R functions, but executes them in an interactive GUI environment (Chang et al., n.d.; Chang and Borges Ribeiro 2018). Fourth, more advanced features for plotting customized SHMs are covered using the Human Brain data set as an example.

2 Getting Started

2.1 Installation

The spatialHeatmap package should be installed from an R (version \(\ge\) 3.6) session with the BiocManager::install command.

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("spatialHeatmap")

2.2 Packages and Documentation

Next, the packages required for running the sample code in this vignette need to be loaded.

library(spatialHeatmap); library(SummarizedExperiment); library(ExpressionAtlas); library(GEOquery)

The following lists the vignette(s) of this package in an HTML browser. Clicking the corresponding name will open this vignette.

browseVignettes('spatialHeatmap')

3 Spatial Heatmaps (SHMs)

3.1 Toy Example

SHMs are plotted with the spatial_hm function. To provide a quick and intuitive overview how these plots are generated, the following uses a generalized toy example where a small vector of random numeric values is generated that are used to color features in an aSVG image. The image chosen for this example is an aSVG depicting the human brain. The corresponding image file ‘homo_sapiens.brain.svg’ is included in this package for testing purposes. The path to this image on a user's system, where spatialHeatmap is installed, can be obtained with the system.file function.

3.1.1 aSVG Image

The following commands obtain the directory of the aSVG collection and the full path to the chosen target aSVG image on a user’s system, respectively.

svg.dir <- system.file("extdata/shinyApp/example", package="spatialHeatmap")
svg.hum <- system.file("extdata/shinyApp/example", 'homo_sapiens.brain.svg', package="spatialHeatmap")

To identify features of interest in annotated aSVG images, the return_feature function can be used. The following searches the aSVG images stored in dir for the query terms ‘lobe’ and ‘homo sapiens’ under the feature and species fields, respectively. The identified matches are returned as a data.frame.

feature.df <- return_feature(feature=c('lobe'), species=c('homo sapiens'), remote=FALSE, dir=svg.dir)
## Accessing features... 
## arabidopsis_thaliana.root_cross.svg, gallus_gallus.svg, homo_sapiens.brain.svg, mus_musculus.male.svg, organ_final.svg, root_cross_final.svg, root_roottip_final.svg, shoot_final.svg, shoot_root_final.svg, us_map_final.svg,
feature.df
##          feature             id                    SVG    parent index index1
## 1 occipital lobe UBERON_0002021 homo_sapiens.brain.svg LAYER_EFO     9      7
## 2  parietal lobe UBERON_0001872 homo_sapiens.brain.svg LAYER_EFO    10      8
## 3  temporal lobe UBERON_0001871 homo_sapiens.brain.svg LAYER_EFO    26     24
fnames <- feature.df[, 1]

3.1.2 Numeric Data

The following example generates a small numeric toy vector, where the data slot contains four numbers and its name slot is populated with the three feature names obtained from the above aSVG image. In addition, a non-matching entry (here ‘notMapped’) is included for demonstration purposes. Note, the numbers are mapped to features via matching names among the numeric vector and the aSVG, respectively. Accordingly, only numbers and features with matching name counterparts can be colored in the aSVG image. Entries without name matches are indicated by a message printed to the R console, here “notMapped”. This behavior can be turned off with verbose=FALSE in the corresponding function call. In addition, a summary of the numeric assay to feature mappings is stored in the result data.frame returned by the spatial_hm function (see below).

my_vec <- sample(1:100, length(unique(fnames))+1)
names(my_vec) <- c(unique(fnames), 'notMapped')
my_vec
## occipital lobe  parietal lobe  temporal lobe      notMapped 
##             18             72             71             65

3.1.3 Plot SHM

Next, the SHM is plotted with the spatial_hm function (Figure 2). Internally, the numbers in my_vec are translated to colors based on the color key assigned to the col.com argument, and then painted onto the corresponding features in the aSVG, where the path to the image file is defined by svg.path=svg.hum. The remaining arguments used here include: ID for defining the title of the plot; ncol for setting the column-wise layout of the plot excluding the feature legend plot on the right; and height for defining the height of the SHM relative to its width. In the given example (Figure 2) only three features in my_vec (‘occipital lobe’, ‘parietal lobe’, and ‘temporal lobe’) have matching entries in the corresponding aSVG.

shm.df <- spatial_hm(svg.path=svg.hum, data=my_vec, ID='toy', ncol=1, height=0.7, sub.title.size=20)
## Syntactically valid column names are made! 
## Enrties not mapped: notMapped
SHM of human brain with toy data. The plots from left to right represent color key, SHM and legend. The colors in the first two plots depict the user provided numeric values, whereas in the legend plot they are used to map the feature labels to the corresponding spatial regions in the image.

Figure 2: SHM of human brain with toy data
The plots from left to right represent color key, SHM and legend. The colors in the first two plots depict the user provided numeric values, whereas in the legend plot they are used to map the feature labels to the corresponding spatial regions in the image.

The named numeric values in my_vec, that have name matches with the features in the chosen aSVG, are stored in the mapped_feature slot.

# The SHM and mapped features are stored in a list
names(shm.df)
## [1] "spatial_heatmap" "mapped_feature"
# Mapped features
shm.df[['mapped_feature']]
##   rowID     featureSVG value
## 1   toy occipital.lobe    18
## 2   toy  parietal.lobe    72
## 3   toy  temporal.lobe    71

3.2 Human Brain

This subsection introduces how to find cell- and tissue-specific assay data in the Expression Atlas database. After choosing a gene expression experiment, the data is downloaded directly into a user's R session. Subsequently, the expression values for selected genes can be plotted onto a chosen aSVG image with or without prior preprocessing steps (e.g. normalization). For querying and downloading expression data from the Expression Atlas database, functions from the ExpressionAtlas package are used (Keays 2019).

3.2.1 Gene Expression Data

The following example searches the Expression Atlas for expression data derived from specific tissues and species of interest, here ‘cerebellum’ and ‘Homo sapiens’, respectively.

all.hum <- searchAtlasExperiments(properties="cerebellum", species="Homo sapiens")

The search result is stored in a DFrame containing accessions matching the above query. For the following sample code, the accession ‘E-GEOD-67196’ from Prudencio et al. (2015) has been chosen, which corresponds to an RNA-Seq profiling experiment of ‘cerebellum’ and ‘frontal cortex’ brain tissue from patients with amyotrophic lateral sclerosis (ALS). Details about the corresponding record can be returned as follows.

all.hum[2, ]
## DataFrame with 1 row and 4 columns
##      Accession      Species                  Type
##    <character>  <character>           <character>
## 1 E-GEOD-67196 Homo sapiens RNA-seq of coding RNA
##                                                                                                                                   Title
##                                                                                                                             <character>
## 1 Transcription profiling by high throughput sequencing of cerebellum and frontal cortex from patients of amyotrophic lateral sclerosis

The getAtlasData function allows to download the chosen RNA-Seq experiment from the Expression Atlas and import it into a RangedSummarizedExperiment object of a user's R session.

rse.hum <- getAtlasData('E-GEOD-67196')[[1]][[1]]
## Downloading Expression Atlas experiment summary from:
##  ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/atlas/experiments/E-GEOD-67196/E-GEOD-67196-atlasExperimentSummary.Rdata
## Successfully downloaded experiment summary object for E-GEOD-67196

The design of the downloaded RNA-Seq experiment is described in the colData slot of rse.hum. The following returns only its first five rows and columns.

colData(rse.hum)[1:5, 1:5]
## DataFrame with 5 rows and 5 columns
##            AtlasAssayGroup     organism   individual  organism_part
##                <character>  <character>  <character>    <character>
## SRR1927019              g1 Homo sapiens  individual1     cerebellum
## SRR1927020              g2 Homo sapiens  individual1 frontal cortex
## SRR1927021              g1 Homo sapiens  individual2     cerebellum
## SRR1927022              g2 Homo sapiens  individual2 frontal cortex
## SRR1927023              g1 Homo sapiens individual34     cerebellum
##                                  disease
##                              <character>
## SRR1927019 amyotrophic lateral sclerosis
## SRR1927020 amyotrophic lateral sclerosis
## SRR1927021 amyotrophic lateral sclerosis
## SRR1927022 amyotrophic lateral sclerosis
## SRR1927023 amyotrophic lateral sclerosis

3.2.2 aSVG Image

The following example shows how to download from the EBI SVG repository an aSVG image that matches the tissues and species assayed in the gene expression data set downloaded in the previous subsection. The return_feature function queries the repository for feature- and species-related keywords, here c('frontal cortex', 'cerebellum') and c('homo sapiens', 'brain'), respectively. To return aSVGs with at least one feature and one species match, the argument keywords.any is set to TRUE by default. When return.all=FALSE, only aSVGs matching the query keywords are returned and saved under dir. Otherwise, all aSVGs are returned regardless of the keywords. To avoid overwriting of existing SVG files, it is recommended to start with an empty target directory, here ~/test. To search a local directory for matching aSVG images, the argument setting remote=FALSE needs to be used, while specifying the path of the corresponding directory under dir. All or only matching features are returned if match.only is set to FALSE or TRUE, respectively.

dir.create('~/test') # Create empty directory
feature.df <- return_feature(feature=c('frontal cortex', 'cerebellum'), species=c('homo sapiens', 'brain'), keywords.any=TRUE, return.all=FALSE, dir='~/test', remote=TRUE, match.only=TRUE, desc=FALSE) # Query aSVGs
feature.df[1:8, ] # Return first 8 rows for checking
unique(feature.df$SVG) # Return all matching aSVGs

To build this vignettes according to the R/Bioconductor package requirements, the following code section uses the aSVG file instance included in the spatialHeatmap package rather than the downloaded instance from the previous example.

feature.df <- return_feature(feature=c('frontal cortex', 'cerebellum'), species=c('homo sapiens', 'brain'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE)
## Accessing features... 
## arabidopsis_thaliana.root_cross.svg, gallus_gallus.svg, homo_sapiens.brain.svg, mus_musculus.male.svg, organ_final.svg, root_cross_final.svg, root_roottip_final.svg, shoot_final.svg, shoot_root_final.svg, us_map_final.svg,

Note, the target tissues frontal cortex and cerebellum are included in both the experimental design slot of the downloaded expression data as well as the annotations of the aSVG. This way these features can be colored in the downstream SHM plots. If necessary users can also change from within R the feature identifiers and names in an aSVG. Details on this utility are provided in the Supplement.

feature.df
##                feature             id                    SVG    parent index
## 1 middle frontal gyrus UBERON_0002702 homo_sapiens.brain.svg LAYER_EFO     8
## 2     cingulate cortex UBERON_0003027 homo_sapiens.brain.svg LAYER_EFO    21
## 3    prefrontal cortex UBERON_0000451 homo_sapiens.brain.svg LAYER_EFO    23
## 4       frontal cortex UBERON_0001870 homo_sapiens.brain.svg LAYER_EFO    24
## 5      cerebral cortex UBERON_0000956 homo_sapiens.brain.svg LAYER_EFO    25
## 6           cerebellum UBERON_0002037 homo_sapiens.brain.svg LAYER_EFO    27
##   index1
## 1      6
## 2     19
## 3     21
## 4     22
## 5     23
## 6     25

Since the Expression Atlas supports the cross-species anatomy ontology, the corresponding UBERON identifiers are included in the id column of the data.frame returned by the above function call of return_feature (Mungall et al. 2012). This ontology is also supported by the rols Bioconductor package (Gatto 2019).

3.2.3 Experimental Design

For organizing experimental designs and downstream plotting purposes, it can be desirable to shorten the text in certain columns of colData. This way one can use the source data for displaying ‘pretty’ sample names in columns and legends of all downstream tables and plots, respectively, in a consistent and automated manner. To achieve this, the following example imports a ‘targets’ file that can be generated and edited by the user in a text or spreadsheet program. In the following example the target file content is used to replace the text in the colData slot with a shortened version.

The following imports a custom target file containing simplified sample labels and experimental design information.

hum.tar <- system.file('extdata/shinyApp/example/target_human.txt', package='spatialHeatmap')
target.hum <- read.table(hum.tar, header=TRUE, row.names=1, sep='\t')

Load custom target data into colData slot.

colData(rse.hum) <- DataFrame(target.hum)

A slice of the simplified colData object is shown below, where the disease column contains now shorter labels than in the original data set. Additional details for generating and using target files in spatialHeatmap are provided in the Supplement of this vignette.

colData(rse.hum)[c(1:3, 41:42), 4:5]
## DataFrame with 5 rows and 2 columns
##             organism_part     disease
##               <character> <character>
## SRR1927019     cerebellum         ALS
## SRR1927020 frontal cortex         ALS
## SRR1927021     cerebellum         ALS
## SRR1927059     cerebellum      normal
## SRR1927060 frontal cortex      normal

3.2.4 Preprocess Assay Data

The actual gene expression data of the downloaded RNA-Seq experiment is stored in the assay slot of rse.hum. Since it contains raw count data, it can be desirable to apply basic preprocessing routines prior to plotting spatial heatmaps. The following shows how to normalize the count data, aggregate replicates and then remove genes with unreliable expression responses. These preprocessing steps are optional and can be skipped if needed. For this, the expression data can be provided to the spatial_hm function directly, where it is important to assign to the sam.factor and/or con.factor arguments the corresponding sample and/or condition column names (Table 2).

For normalizing raw count data from RNA-Seq experiments, the norm_data function can be used. It supports the following pre-existing functions from widely used packages for analyzing count data in the next generation sequencing (NGS) field: calcNormFactors (CNF) from edgeR (McCarthy et al. 2012); as well as estimateSizeFactors (EST), varianceStabilizingTransformation (VST), and rlog from DESeq2 (Love, Huber, and Anders 2014). The argument norm.fun specifies one of the four internal normalizing methods: CNF, EST, VST, and rlog. If norm.fun='none', no normalization is applied. The arguments for each normalizing function are provided via a parameter.list, which is a list with named slots. For example, norm.fun='ESF' and parameter.list=list(type='ratio') is equivalent to estimateSizeFactors(object, type='ratio'). If paramter.list=NULL, the default arguments are used by the normalizing function assigned to norm.fun. For additional details, users want to consult the help file of the norm_data function by typing ?norm_data in the R console.

The following example uses the ESF normalization option. This method has been chosen mainly due to its good time performance.

se.nor.hum <- norm_data(data=rse.hum, norm.fun='ESF', data.trans='log2')
## Normalising: ESF 
##    type 
## "ratio"

Replicates are aggregated with the aggr_rep function, where the summary statistics can be chosen under the aggr argument (e.g. aggr='mean'). The columns specifying replicates can be assigned to the sam.factor and con.factor arguments corresponding to samples and conditions, respectively. For tracking, the corresponding sample/condition labels are used as column titles in the aggregated assay instance, where they are concatenated with a double underscore as separator. In addition, the corresponding rows in the colData slot are collapsed accordingly.

se.aggr.hum <- aggr_rep(data=se.nor.hum, sam.factor='organism_part', con.factor='disease', aggr='mean')
## Syntactically valid column names are made!
assay(se.aggr.hum)[1:3, ]
##                 cerebellum__ALS frontal.cortex__ALS cerebellum__normal
## ENSG00000000003        7.024054            7.091484           6.406157
## ENSG00000000005        0.000000            1.540214           0.000000
## ENSG00000000419        7.866582            8.002549           8.073264
##                 frontal.cortex__normal
## ENSG00000000003               7.004446
## ENSG00000000005               1.403110
## ENSG00000000419               7.955709

To remove unreliable expression measures, filtering can be applied. The following example eliminates genes with expression values larger than 5 (log2 space) in at least 1% of all samples (pOA=c(0.01, 5)), while retaining genes with a coefficient of variance (CV) between 0.30 and 100 (CV=c(0.30, 100)).

se.fil.hum <- filter_data(data=se.aggr.hum, sam.factor='organism_part', con.factor='disease', pOA=c(0.01, 5), CV=c(0.3, 100), dir=NULL)
## Syntactically valid column names are made!

To inspect the results, the following returns three selected rows of the fully preprocessed data matrix (Table 1).

assay(se.fil.hum)[c(5, 733:734), ]

Table 1: Slice of fully preprocessed expression matrix.
cerebellum__ALS frontal.cortex__ALS cerebellum__normal frontal.cortex__normal
ENSG00000006047 1.134172 5.2629629 0.5377534 5.3588310
ENSG00000268433 5.324064 0.3419665 3.4780744 0.1340332
ENSG00000268555 5.954572 2.6148548 4.9349736 2.0351776

3.2.5 SHM: Single Gene

The preprocessed expression values for any gene in the assay slot of se.fil.hum can be plotted as a SHM. The following uses gene ENSG00000268433 as an example. The chosen aSVG is a depiction of the human brain where the assayed featured are colored by the corresponding expression values in se.fil.hum.

shm.df <- spatial_hm(svg.path=svg.hum, data=se.fil.hum, ID=c('ENSG00000268433'), height=0.6, legend.r=1.3)
SHM of human brain. Only cerebellum and frontal cortex are colored, because they are present in both the aSVG and the expression data. The legend plot on the right maps the feature labels to the corresponding spatial regions in the image.

Figure 3: SHM of human brain
Only cerebellum and frontal cortex are colored, because they are present in both the aSVG and the expression data. The legend plot on the right maps the feature labels to the corresponding spatial regions in the image.

The plotting instructions of the SHM along with the corresponding mapped features are stored in a list named shm.df. Its components can be accessed as follows.

names(shm.df)
## [1] "spatial_heatmap" "mapped_feature"
# Mapped features
shm.df[['mapped_feature']]
##             rowID     featureSVG condition     value
## 1 ENSG00000268433     cerebellum       ALS 5.3240638
## 2 ENSG00000268433 frontal.cortex       ALS 0.3419665
## 3 ENSG00000268433     cerebellum    normal 3.4780744
## 4 ENSG00000268433 frontal.cortex    normal 0.1340332

In the above example, the normalized expression values of gene ENSG00000268433 are colored in the frontal cortex and cerebellum, while the different conditions, here normal and ALS, are given in separate SHMs plotted next to each other (Figure 3). The color and feature mappings are defined by the corresponding color key and legend plot on the left and right, respectively.

3.2.6 SHM: Multiple Genes

SHMs for multiple genes can be plotted by providing the corresponding gene IDs under the ID argument as a character vector. The spatial_hm function will then sequentially arrange the SHMs for each gene in a single composite plot. To facilitate comparisons of expression values across genes and/or conditions, the lay.shm parameter can be assigned 'gene' or 'con', respectively. For instance, in Figure 4 the SHMs of the genes ENSG00000268433 and ENSG00000006047 are organised by condition in a horizontal view. This functionality is particularly useful when comparing gene families.

spatial_hm(svg.path=svg.hum, data=se.fil.hum, ID=c('ENSG00000268433', 'ENSG00000006047'), lay.shm='con', width=1, height=1, legend.r=1.5)
SHMs of two genes. The subplots are organised by "condition" through `lay.shm` argument.

Figure 4: SHMs of two genes
The subplots are organised by “condition” through lay.shm argument.

3.2.7 SHM: Customization

To provide a high level of flexibility, the spatial_hm contains many arguments. An overview of the argument names and their utility is provide in Table 2.


Table 2: Argument list of ‘spatial_hm’.
argument description
svg.path Path of aSVG
data Input data of SummarizedExperiment (SE), data frame, or vector
sam.factor Applies to SE. Column name of sample replicates in colData slot. Default is NULL
con.factor Applies to SE. Column name of condition replicates in colData slot. Default is NULL
ID A character vector of row items for plotting spatial heatmaps
col.com A character vector of color components for building colour scale. Default is c(‘purple’, ‘yellow’, ‘blue’)
col.bar ‘selected’ or ‘all’, the former means use values of ID to build the colour scale while the latter use all values in data. Default is ‘selected’.
bar.width A numeric of colour bar width. Default is 0.7
data.trans ‘log2’, ‘exp2’, or NULL, ‘log2’ transforms data to log2 scale for plotting while ‘exp2’ to 2-base exponent. Default is NULL, no transformation.
tis.trans A vector of aSVG features to be transparent. Default is NULL.
width, height Two numerics of width and height of spatial heatmap plots repsectively. Default is 1, 1.
legend.r The ratio aspect (width to height) of legend plot. Default is 1.
sub.title.size The title size of each spatial heatmap subplot. Default is 11.
lay.shm ‘gen’ or ‘con’, applies to multiple genes or conditions respectively. ‘gen’ means spatial heatmaps are organised by genes while ‘con’ organised by conditions. Default is ‘gen’
ncol The total column number of spatial heatmaps, not including legend plot. Default is 2.
sam.legend ‘identical’, ‘all’, or a vector of samples/features in aSVG to show in legend plot. ‘identical’ only shows matching features while ‘all’ shows all features.
legend.ncol, legend.nrow Two numbers of columns and rows of legend keys respectively. Default is NULL, NULL, since they are automatically set.
legend.position the position of legend keys (‘none’, ‘left’, ‘right’,‘bottom’, ‘top’), or two-element numeric vector. Default is ‘bottom’.
legend.direction Layout of keys in legends (‘horizontal’ or ‘vertical’). Default is NULL, since it is automatically set.
legend.key.size, legend.label.size The size of legend keys and labels respectively. Default is 0.5 and 8 respectively.
line.size, line.color The size and colour of all plogyon outlines respectively. Default is 0.2 and ‘grey70’ respectively.
verbose TRUE or FALSE. Default is TRUE and the aSVG features not mapped are printed to R console.

3.3 Mouse Organs

This section generates an SHM plot for mouse data from the Expression Atlas. The code components are very similar to the previous Human Brain example. For brevity, the corresponding text explaining the code has been reduced to a minimum.

3.3.1 Gene Expression Data

The chosen mouse RNA-Seq data compares tissue level gene expression across mammalian species (Merkin et al. 2012). The following searches the Expression Atlas for expression data from ‘heart’ and ‘Mus musculus’.

all.mus <- searchAtlasExperiments(properties="heart", species="Mus musculus")
## Searching for Expression Atlas experiments matching your query ...
## Query successful.
## Found 67 experiments matching your query.

Among the many matching entries, accession ‘E-MTAB-2801’ will be downloaded.

all.mus[7, ]
## DataFrame with 1 row and 4 columns
##     Accession      Species                  Type
##   <character>  <character>           <character>
## 1 E-MTAB-2801 Mus musculus RNA-seq of coding RNA
##                                           Title
##                                     <character>
## 1 Strand-specific RNA-seq of nine mouse tissues
rse.mus <- getAtlasData('E-MTAB-2801')[[1]][[1]]
## Downloading Expression Atlas experiment summary from:
##  ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/atlas/experiments/E-MTAB-2801/E-MTAB-2801-atlasExperimentSummary.Rdata
## Successfully downloaded experiment summary object for E-MTAB-2801

The design of the downloaded RNA-Seq experiment is described in the colData slot of rse.mus. The following returns only its first three rows.

colData(rse.mus)[1:3, ]
## DataFrame with 3 rows and 4 columns
##           AtlasAssayGroup     organism organism_part      strain
##               <character>  <character>   <character> <character>
## SRR594393              g7 Mus musculus         brain      DBA/2J
## SRR594394             g21 Mus musculus         colon      DBA/2J
## SRR594395             g13 Mus musculus         heart      DBA/2J

3.3.2 aSVG Image

The following example shows how to download from the EBI SVG repository an aSVG image that matches the tissues and species assayed in the gene expression data set downloaded in the previous subsection. As before the image is saved to a directory named ~/test.

if (!dir.exists('~/test')) dir.create('~/test')
feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('Mus musculus'), keywords.any=TRUE, return.all=FALSE, dir='~/test', remote=TRUE, match.only=FALSE)

To build this vignettes according to the R/Bioconductor package requirements, the following code section uses the aSVG file instance included in the spatialHeatmap package rather than the downloaded instance from the previous example.

feature.df <- return_feature(feature=c('heart', 'kidney'), species=NULL, keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE) 
## Accessing features... 
## arabidopsis_thaliana.root_cross.svg, gallus_gallus.svg, homo_sapiens.brain.svg, mus_musculus.male.svg, organ_final.svg, root_cross_final.svg, root_roottip_final.svg, shoot_final.svg, shoot_root_final.svg, us_map_final.svg,

Return the names of the matching aSVG files.

unique(feature.df$SVG)
## [1] "gallus_gallus.svg"     "mus_musculus.male.svg"

The following first selects mus_musculus.male.svg as target aSVG, then returns the first three rows of the resulting feature.df, and finally prints the unique set of all aSVG features.

feature.df <- subset(feature.df, SVG=='mus_musculus.male.svg')
feature.df[1:3, ]
##     feature             id                   SVG        parent index index1
## 10   kidney UBERON_0002113 mus_musculus.male.svg     LAYER_EFO    14     12
## 11    heart UBERON_0000948 mus_musculus.male.svg     LAYER_EFO    51     49
## 12 path4204       path4204 mus_musculus.male.svg LAYER_OUTLINE     1      1
unique(feature.df[, 1])
##  [1] "kidney"                    "heart"                    
##  [3] "path4204"                  "aorta"                    
##  [5] "circulatory system"        "blood vessel"             
##  [7] "brown adipose tissue"      "white adipose tissue"     
##  [9] "skin"                      "stomach"                  
## [11] "duodenum"                  "pancreas"                 
## [13] "spleen"                    "adrenal gland"            
## [15] "colon"                     "small intestine"          
## [17] "caecum"                    "jejunum"                  
## [19] "ileum"                     "esophagus"                
## [21] "gall bladder"              "parotid gland"            
## [23] "submandibular gland"       "lymph node"               
## [25] "parathyroid gland"         "tongue"                   
## [27] "Peyer’s patch"             "prostate gland"           
## [29] "vas deferens"              "epididymis"               
## [31] "testis"                    "seminal vesicle"          
## [33] "penis"                     "urinary bladder"          
## [35] "thymus"                    "femur"                    
## [37] "bone marrow"               "cartilage"                
## [39] "quadriceps femoris"        "spinal cord"              
## [41] "lung"                      "diaphragm"                
## [43] "peripheral nervous system" "trachea"                  
## [45] "hindlimb"                  "trigeminal nerve"         
## [47] "eye"                       "sciatic nerve"            
## [49] "intestinal mucosa"         "liver"                    
## [51] "brain"                     "skeletal muscle"

Obtain path of target aSVG on user system.

svg.mus <- system.file("extdata/shinyApp/example", "mus_musculus.male.svg", package="spatialHeatmap")

3.3.3 Experimental Design

Import of custom target file defining simplified sample labels and experimental design. The following imports a sample target file that is included in this package. To inspect its content, the first three rows of the target file are printed to the screen.

mus.tar <- system.file('extdata/shinyApp/example/target_mouse.txt', package='spatialHeatmap')
target.mus <- read.table(mus.tar, header=TRUE, row.names=1, sep='\t')
target.mus[1:3, ]
##           AtlasAssayGroup     organism organism_part strain
## SRR594393              g7 Mus musculus         brain DBA.2J
## SRR594394             g21 Mus musculus         colon DBA.2J
## SRR594395             g13 Mus musculus         heart DBA.2J
unique(target.mus[, 3])
## [1] "brain"           "colon"           "heart"           "kidney"         
## [5] "liver"           "lung"            "skeletal muscle" "spleen"         
## [9] "testis"

Load custom target data into colData slot.

colData(rse.mus) <- DataFrame(target.mus)

3.3.4 Preprocess Assay Data

The raw RNA-Seq count are preprocessed with the following steps: (1) normalization, (2) aggregation of replicates, and (3) filtering of reliable expression data. The details of these steps are explained in the previous sub-section using data from human.

se.nor.mus <- norm_data(data=rse.mus, norm.fun='ESF', data.trans='log2') # Normalization
## Normalising: ESF 
##    type 
## "ratio"
se.aggr.mus <- aggr_rep(data=se.nor.mus, sam.factor='organism_part', con.factor='strain', aggr='mean') # Aggregation of replicates
## Syntactically valid column names are made!
se.fil.mus <- filter_data(data=se.aggr.mus, sam.factor='organism_part', con.factor='strain', pOA=c(0.01, 5), CV=c(0.6, 100), dir=NULL) # Filtering of genes with low counts and variance 
## Syntactically valid column names are made!

3.3.5 SHM: Transparency

The pre-processed expression data for gene ‘ENSMUSG00000000263’ is plotted in form of an SHM. In this case the plot includes expression data for 8 tissues across 3 mouse strains.

spatial_hm(svg.path=svg.mus, data=se.fil.mus, ID=c('ENSMUSG00000000263'), height=0.5, legend.r=1.1, sub.title.size=9, ncol=3, tis.trans=c('skeletal muscle'), legend.nrow=4, line.size=0.2, line.color='grey70')
SHM of mouse organs. This is a multiple-layer image where the polygon of the 'skeletal muscle' is set transparent to expose 'lung' and 'heart'.

Figure 5: SHM of mouse organs
This is a multiple-layer image where the polygon of the ‘skeletal muscle’ is set transparent to expose ‘lung’ and ‘heart’.

The SHM plots in Figures 5 and 6 demonstrate the usage of the transparency feature via the tis.trans parameter. The corresponding mouse organ aSVG image includes overlapping tissue layers. In this case the skelectal muscle layer partially overlaps with lung and heart tissues. To view lung and heart in Figure 5, the skelectal muscle tissue is set transparent with tis.trans=c('skeletal muscle'). To view in the same aSVG the skeletal muscle tissue instead, tis.trans is assigned NULL for generating the SHM plot of Figure 6.

To fine control the visual effects in feature rich aSVGs, the line.size and line.color parameters are useful. This way one can adjust the thickness and color of complex polygon structures.

spatial_hm(svg.path=svg.mus, data=se.fil.mus, ID=c('ENSMUSG00000000263'), height=0.5, legend.r=1.1, sub.title.size=9, ncol=3, tis.trans=NULL, legend.ncol=2, line.size=0.2, line.color='grey70')
SHM of mouse organs. This is a multiple-layer image where the view onto 'lung' and 'heart' is obstructed by displaying the 'skeletal muscle' tissue.

Figure 6: SHM of mouse organs
This is a multiple-layer image where the view onto ‘lung’ and ‘heart’ is obstructed by displaying the ‘skeletal muscle’ tissue.

3.4 Chicken Organs

This section generates an SHM plot for chicken data from the Expression Atlas. The code components are very similar to the Human Brain example. For brevity, the corresponding text explaining the code has been reduced to a minimum.

3.4.1 Gene Expression Data

The chosen chicken RNA-Seq experiment compares the developmental changes across nine time points of seven organs (Cardoso-Moreira et al. 2019).

The following searches the Expression Atlas for expression data from ‘heart’ and ‘gallus’.

all.chk <- searchAtlasExperiments(properties="heart", species="gallus")
## Searching for Expression Atlas experiments matching your query ...
## Query successful.
## Found 3 experiments matching your query.

Among the matching entries, accession ‘E-MTAB-6769’ will be downloaded.

all.chk[3, ]
## DataFrame with 1 row and 4 columns
##     Accession       Species                  Type
##   <character>   <character>           <character>
## 1 E-MTAB-6769 Gallus gallus RNA-seq of coding RNA
##                                                                  Title
##                                                            <character>
## 1 Chicken RNA-seq time-series of the development of seven major organs
rse.chk <- getAtlasData('E-MTAB-6769')[[1]][[1]]
## Downloading Expression Atlas experiment summary from:
##  ftp://ftp.ebi.ac.uk/pub/databases/microarray/data/atlas/experiments/E-MTAB-6769/E-MTAB-6769-atlasExperimentSummary.Rdata
## Successfully downloaded experiment summary object for E-MTAB-6769

The design of the downloaded RNA-Seq experiment is described in the colData slot of rse.chk. The following returns only its first three rows.

colData(rse.chk)[1:3, ]
## DataFrame with 3 rows and 8 columns
##            AtlasAssayGroup      organism         strain           genotype
##                <character>   <character>    <character>        <character>
## ERR2576379              g1 Gallus gallus Red Junglefowl wild type genotype
## ERR2576380              g1 Gallus gallus Red Junglefowl wild type genotype
## ERR2576381              g2 Gallus gallus Red Junglefowl wild type genotype
##            developmental_stage         age         sex organism_part
##                    <character> <character> <character>   <character>
## ERR2576379              embryo      10 day      female         brain
## ERR2576380              embryo      10 day      female         brain
## ERR2576381              embryo      10 day      female    cerebellum

3.4.2 aSVG Image

The following example shows how to download from the EBI SVG repository an aSVG image that matches the tissues and species assayed in the gene expression data set downloaded in the previous subsection. As before the image is saved to a directory named ~/test.

# Make an empty directory "~/test" if not exist.
if (!dir.exists('~/test')) dir.create('~/test')
# Query aSVGs.
feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('gallus'), keywords.any=TRUE, return.all=FALSE, dir='~/test', remote=TRUE, match.only=FALSE)

To build this vignettes according to the R/Bioconductor package requirements, the following code section uses the aSVG file instance included in the spatialHeatmap package rather than the downloaded instance from the previous example.

feature.df <- return_feature(feature=c('heart', 'kidney'), species=c('gallus'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE)
## Accessing features... 
## arabidopsis_thaliana.root_cross.svg, gallus_gallus.svg, homo_sapiens.brain.svg, mus_musculus.male.svg, organ_final.svg, root_cross_final.svg, root_roottip_final.svg, shoot_final.svg, shoot_root_final.svg, us_map_final.svg,
feature.df
##                 feature              id               SVG        parent index
## 1                 heart  UBERON_0000948 gallus_gallus.svg     LAYER_EFO     4
## 2                kidney  UBERON_0002113 gallus_gallus.svg     LAYER_EFO     5
## 3       chicken_outline chicken_outline gallus_gallus.svg LAYER_OUTLINE     1
## 4                 brain  UBERON_0000955 gallus_gallus.svg     LAYER_EFO     3
## 5                 liver  UBERON_0002107 gallus_gallus.svg     LAYER_EFO     6
## 6 skeletal muscle organ  UBERON_0014892 gallus_gallus.svg     LAYER_EFO     7
## 7                 colon  UBERON_0001155 gallus_gallus.svg     LAYER_EFO     8
## 8                spleen  UBERON_0002106 gallus_gallus.svg     LAYER_EFO     9
## 9                  lung  UBERON_0002048 gallus_gallus.svg     LAYER_EFO    10
##   index1
## 1      2
## 2      3
## 3      1
## 4      1
## 5      4
## 6      5
## 7      6
## 8      7
## 9      8

Obtain path of target aSVG on user system.

svg.chk <- system.file("extdata/shinyApp/example", "gallus_gallus.svg", package="spatialHeatmap")

3.4.3 Experimental Design

Import of custom target file defining simplified sample labels and experimental design. The following imports a sample target file that is included in this package. To inspect its content, the first three rows of the target file are printed to the screen.

chk.tar <- system.file('extdata/shinyApp/example/target_chicken.txt', package='spatialHeatmap')
target.chk <- read.table(chk.tar, header=TRUE, row.names=1, sep='\t')
target.chk[1:3, ]
##            AtlasAssayGroup      organism         strain           genotype
## ERR2576379              g1 Gallus gallus Red Junglefowl wild type genotype
## ERR2576380              g1 Gallus gallus Red Junglefowl wild type genotype
## ERR2576381              g2 Gallus gallus Red Junglefowl wild type genotype
##            developmental_stage   age    sex organism_part
## ERR2576379              embryo day10 female         brain
## ERR2576380              embryo day10 female         brain
## ERR2576381              embryo day10 female    cerebellum

Load custom target data into colData slot.

colData(rse.chk) <- DataFrame(target.chk)

All samples used for plotting SHMs.

unique(colData(rse.chk)[, 'organism_part'])
## [1] "brain"      "cerebellum" "heart"      "kidney"     "ovary"     
## [6] "testis"     "liver"

Return conditions considered for plotting downstream SHM.

unique(colData(rse.chk)[, 'age'])
## [1] "day10"  "day12"  "day14"  "day17"  "day0"   "day155" "day35"  "day7"  
## [9] "day70"

3.4.4 Preprocess Assay Data

The raw RNA-Seq count are preprocessed with the following steps: (1) normalization, (2) aggregation of replicates, and (3) filtering of reliable expression data. The details of these steps are explained in the above sub-section using data from human.

se.nor.chk <- norm_data(data=rse.chk, norm.fun='ESF', data.trans='log2') # Normalization
## Normalising: ESF 
##    type 
## "ratio"
se.aggr.chk <- aggr_rep(data=se.nor.chk, sam.factor='organism_part', con.factor='age', aggr='mean') # Replicate agggregation using mean 
se.fil.chk <- filter_data(data=se.aggr.chk, sam.factor='organism_part', con.factor='age', pOA=c(0.01, 5), CV=c(0.6, 100), dir=NULL) # Filtering of genes with low counts and varince

3.4.5 SHM: Time Series

The expression profile for gene ENSGALG00000006346 is plotted across nine time points in four organs in form of a composite SHM with 9 panels. Their layout in three columns is controlled with the argument setting ncol=3.

jianhai-reply: reordered.2 The order of the time points in Figure 7 should be by time. Right now it is rather random.

spatial_hm(svg.path=svg.chk, data=se.fil.chk, ID='ENSGALG00000006346', legend.r=1.5, sub.title.size=9, ncol=3, legend.nrow=2)
## Enrties not mapped: cerebellum, ovary, testis
Time course of chicken organs. The SHM shows the expression profile of a single gene across nine time points and four organs.

Figure 7: Time course of chicken organs
The SHM shows the expression profile of a single gene across nine time points and four organs.

3.5 Arabidopsis Shoot

This section generates an SHM for Arabidopsis thaliana with gene expression data from the Affymetrix microarray technology. The chosen experiment used ribosome-associated mRNAs from several cell populations of shoots and roots that were exposed to hypoxia stress (Mustroph et al. 2009). In this case the expression data will be downloaded from GEO with utilites from the GEOquery package (S. Davis and Meltzer 2007) and the preprocessing routines will be specific to the Affymetrix technology. The remaining code components for generating SHMs are very similar to the previous examples. For brevity, the text in this section explains mainly the steps that are specific to this data set.

3.5.1 Gene Expression Data

The GSE14502 data set is downloaded with the getGEO function from the GEOquery package. Intermediately, the expression data is stored in an ExpressionSet container and then converted to a SummarizedExperiment object.

gset <- getGEO("GSE14502", GSEMatrix=TRUE, getGPL=TRUE)[[1]]
## Found 1 file(s)
## GSE14502_series_matrix.txt.gz
## Using locally cached version: /tmp/RtmpfvRL1j/GSE14502_series_matrix.txt.gz
## Parsed with column specification:
## cols(
##   .default = col_double(),
##   ID_REF = col_character()
## )
## See spec(...) for full column specifications.
## Using locally cached version of GPL198 found here:
## /tmp/RtmpfvRL1j/GPL198.soft
## Warning: 64 parsing failures.
##   row     col           expected    actual         file
## 22747 SPOT_ID 1/0/T/F/TRUE/FALSE --Control literal data
## 22748 SPOT_ID 1/0/T/F/TRUE/FALSE --Control literal data
## 22749 SPOT_ID 1/0/T/F/TRUE/FALSE --Control literal data
## 22750 SPOT_ID 1/0/T/F/TRUE/FALSE --Control literal data
## 22751 SPOT_ID 1/0/T/F/TRUE/FALSE --Control literal data
## ..... ....... .................. ......... ............
## See problems(...) for more details.
se.sh <- as(gset, "SummarizedExperiment")

The gene symbol identifiers are extracted from the rowData component and used as row names. Similarly, one can work with AGI identifiers by providing below AGI under Gene.Symbol.

rownames(se.sh) <- make.names(rowData(se.sh)[, 'Gene.Symbol'])

The following returns a slice of the experimental design stored in the colData slot. Both the samples and conditions are contained in the title column. The samples include promoters (pGL2, pCO2, pSCR, pWOL, p35S), tissues and organs (root atrichoblast epidermis, root cortex meristematic zone, root endodermis, root vasculature, root_total and shoot_total); and the conditions are control and hypoxia.

colData(se.sh)[60:63, 1:4]
## DataFrame with 4 rows and 4 columns
##                              title geo_accession                status
##                        <character>   <character>           <character>
## GSM362227  shoot_hypoxia_pGL2_rep1     GSM362227 Public on Oct 12 2009
## GSM362228  shoot_hypoxia_pGL2_rep2     GSM362228 Public on Oct 12 2009
## GSM362229 shoot_control_pRBCS_rep1     GSM362229 Public on Oct 12 2009
## GSM362230 shoot_control_pRBCS_rep2     GSM362230 Public on Oct 12 2009
##           submission_date
##               <character>
## GSM362227     Jan 21 2009
## GSM362228     Jan 21 2009
## GSM362229     Jan 21 2009
## GSM362230     Jan 21 2009

3.5.2 aSVG Image

In this example, the aSVG image has been regenerated in Inkscape from the corresponding figure in Mustroph et al. (2009). The resulting custom figure has been included as a sample aSVG file in the spatialHeatmap package. Detailed instructions for generating custom aSVG images in Inkscape are provided in the SVG tutorial.

The annotations in the corresponding aSVG file located under svg.dir can be queried with the return_features function.

feature.df <- return_feature(feature=c('pGL2', 'pRBCS'), species=c('shoot'), keywords.any=TRUE, return.all=FALSE, dir=svg.dir, remote=FALSE, match.only=FALSE)
## Accessing features... 
## arabidopsis_thaliana.root_cross.svg, gallus_gallus.svg, homo_sapiens.brain.svg, mus_musculus.male.svg, organ_final.svg, root_cross_final.svg, root_roottip_final.svg, shoot_final.svg, shoot_root_final.svg, us_map_final.svg,

The unique set of the matching aSVG files can be returned as follows.

unique(feature.df$SVG)
## [1] "shoot_final.svg"      "shoot_root_final.svg"

The aSVG file shoot_final.svg is chosen to generate the SHM in this section.

feature.df <- subset(feature.df, SVG=='shoot_final.svg')
feature.df[1:3, ]
##       feature          id             SVG    parent index index1
## 1  shoot_pGL2  shoot_pGL2 shoot_final.svg container     2      2
## 2 shoot_pRBCS shoot_pRBCS shoot_final.svg container     3      3
## 3        g258        g258 shoot_final.svg container     1      1

Obtain full path of target aSVG on user system.

svg.sh <- system.file("extdata/shinyApp/example", "shoot_final.svg", package="spatialHeatmap")

3.5.3 Experimental Design

The following imports a sample target file that is included in this package. To inspect its content, four selected rows of this target file are printed to the screen.

sh.tar <- system.file('extdata/shinyApp/example/target_arab.txt', package='spatialHeatmap')
target.sh <- read.table(sh.tar, header=TRUE, row.names=1, sep='\t')
target.sh[60:63, ]
##                           col.name     samples conditions
## shoot_hypoxia_pGL2_rep1  GSM362227  shoot_pGL2    hypoxia
## shoot_hypoxia_pGL2_rep2  GSM362228  shoot_pGL2    hypoxia
## shoot_control_pRBCS_rep1 GSM362229 shoot_pRBCS    control
## shoot_control_pRBCS_rep2 GSM362230 shoot_pRBCS    control

Return all samples present in target file.

unique(target.sh[, 'samples'])
##  [1] "root_total"      "root_p35S"       "root_pSCR"       "root_pSHR"      
##  [5] "root_pWOL"       "root_pGL2"       "root_pSUC2"      "root_pSultr2.2" 
##  [9] "root_pCO2"       "root_pPEP"       "root_pRPL11C"    "shoot_total"    
## [13] "shoot_p35S"      "shoot_pGL2"      "shoot_pRBCS"     "shoot_pSUC2"    
## [17] "shoot_pSultr2.2" "shoot_pCER5"     "shoot_pKAT1"

Return all conditions present in target file.

unique(target.sh[, 'conditions'])
## [1] "control" "hypoxia"

Load custom target data into colData slot.

colData(se.sh) <- DataFrame(target.sh)

3.5.4 Preprocess Assay Data

The downloaded GSE14502 data set has already been normalized with the RMA algorithm (Gautier et al. 2004). Thus, the pre-processing steps can be restricted to the aggregation of replicates and filtering of reliably expressed genes. For the latter, the following code will retain genes with expression values larger than 6 (log2 space) in at least 3% of all samples (pOA=c(0.03, 6)), and with a coefficient of variance (CV) between 0.30 and 100 (CV=c(0.30, 100)).

se.aggr.sh <- aggr_rep(data=se.sh, sam.factor='samples', con.factor='conditions', aggr='mean') # Replicate agggregation using mean
se.fil.arab <- filter_data(data=se.aggr.sh, sam.factor='samples', con.factor='conditions', pOA=c(0.03, 6), CV=c(0.30, 100), dir=NULL) # Filtering of genes with low intensities and variance

3.5.5 SHM: Microarray Example

The expression profile for the HRE2 gene is plotted for control and hypoxia treatment across six cell types (Figure 8).

spatial_hm(svg.path=svg.sh, data=se.fil.arab, ID=c("HRE2"), height=0.6, legend.nrow=3, legend.r=1.3, legend.key.size=0.3)
## Enrties not mapped: root_total, root_p35S, root_pSCR, root_pSHR, root_pWOL, root_pGL2, root_pSUC2, root_pSultr2.2, root_pCO2, root_pPEP, root_pRPL11C, shoot_total, shoot_p35S
SHM of Arabidopsis shoots. The expression profile of the HRE2 gene is plotted for control and hypoxia treatment across six cell types.

Figure 8: SHM of Arabidopsis shoots
The expression profile of the HRE2 gene is plotted for control and hypoxia treatment across six cell types.

4 Matrix Heatmaps

SHMs are a visualization approach suitable for comparing the expression profiles of single genes or a small number of them across cell types and conditions. To also support analyses across larger number of genes, spatialHeatmap provides utilities for identifying for a gene of interest nearest neighbor genes that share similar expression profiles. This is achieved by identifying clusters and network modules using hierarchical clustering and network analysis. These approaches are described in this and the following sections, respectively.

Adjacency Matrix and Module Identification3 Why this title if there are no additional ones in this section. Is this an orphan leftover from somewhere else?

[ThG-Comment: this section needs major improvements. The intent was to identify for a target gene nearest neighbors based on some similarity metric for gene expression profiles (e.g. correlation), and then hierarchically cluster them and visualize the result in a heatmap. Instead you are subjecting the entire expression matrix to clustering/WGCNA and then plot the target gene in the context of a single cluster/module. This is hugely inefficient compared to doing it only for the nearest neighbors instead. Just think about what will happen if a user imports an expression matrix for all transcripts from human. With your approach they will not be able to complete this step since they will run out of memory on their system.]

The modules are identified by adj_mod. It first computes an adjacency matrix4 You need to describe how this adjency matrix is generated and what it represents. It also needs to mention what type of disance/correlation method is used initially to generate the downstream matrix. on the gene expression matrix then hierarchically clusters the adjacency matrix by using WGCNA (Langfelder and Horvath 2008)5 Unclear for what you need WGCNA here. I thought this section uses hierarchical clustering. The latter is certainly used by WGCNA but it remains a mystery what exactly you are doing here. I would expect to use here only hierarchical clustering, e.g. with flashClust. WGCNA seems more suitable for the next section.] and flashClust (Langfelder and Horvath 2012). The clutersing includes 4 alternative sensitivity levels (ds=0, 1, 2, or 3).6 What is the meaning of the sensitivity levels, how are they generated and how should the user interpret them? From 3 to 0, the sensitivity decreases and results in less modules with larger sizes. Since the interactive network functionality performs better on smaller modules, only ds of 3 and 2 are used. There is another parameter type for module identification: signed and unsinged. The former means both positive and negative adjacency between genes are used while the latter takes the absolute values of negative adjacency7 Without defining how adjacency is defined here readers will not be able to understand what was done here, especially since this section is mainly about hierarchical clustering..

The function adj_mod returns a list containing an adjacency matrix and a data frame of module assignment. It is domenstrated on the Arabidopsis Shoot data.

adj.mod <- adj_mod(data=se.fil.arab)

The adjacency matrix is a measure of co-expression similarity between genes, where larger value denotes more similarity.

adj.mod[['adj']][1:3, 1:3]
##           ndhA      petL      psaJ
## ndhA 1.0000000 0.5374043 0.6088355
## petL 0.5374043 1.0000000 0.7779227
## psaJ 0.6088355 0.7779227 1.0000000

The module assignment is a data frame. The first column is ds=2 while the second is ds=3. The numbers in each column are module labels with “0” meaning genes not assigned to any modules.

adj.mod[['mod']][1:3, ]
##      2 3
## ndhA 1 0
## petL 1 0
## psaJ 1 0

The matrix heatmap is implemented in function matrix_hm with 2 modes provided: static or interactive. Figure 9 is the static mode on gene HRE2. Setting static=FALSE launches the interactive mode, where users can zoom in and out by drawing a rectangle and by double clicking the heatmap, respectively.

matrix_hm(geneID="HRE2", data=se.fil.arab, adj.mod=adj.mod, angleCol=80, angleRow=35, cexRow=0.8, cexCol=0.8, margin=c(10, 6), static=TRUE, arg.lis1=list(offsetRow=0.1, offsetCol=0.1))
Matrix Heatmap. Rows are genes and columns are samples. The input gene is tagged by 2 black lines.

Figure 9: Matrix Heatmap
Rows are genes and columns are samples. The input gene is tagged by 2 black lines.

In Figure 9, the target gene is displayed in the gene module it belongs to, which is indicated by 2 black lines. The rows and columns are sorted by hierarchical clustering dendrograms. The expression matrix of this module is visualised without being scaled (scale="no"). It can be seen that the expression levels of this module is overall much higher in hypoxia than control, and therefore it could potentially be used to infer the hypoxia response mechanism in Arabidopsis.

5 Network Graphs

[ThG-Comment: both the previous section and this section need to clearly explain what is done to arrive at a given network module and what it represents. Once this is done I will edit the text.]

The same target gene and module from matrix heatmap can also be displayed as a network. Similarly, the network can be dispayed in static or interactive mode.

Setting static=TRUE launches the static network. In Figure 10 Nodes are genes and edges are adjacencies between genes. The thicker edge denotes higher adjacency (co-expression similarity) while larger node indicates higher gene connectivity (sum of a gene's adjacency with all its direct neighbours). The target gene is labeled by ’_selected’.

network(geneID="HRE2", data=se.fil.arab, adj.mod=adj.mod, adj.min=0.75, vertex.label.cex=1.2, vertex.cex=2, static=TRUE)
Static network. Node size denotes gene connectivity while edge thickness stands for co-expression similarity.

Figure 10: Static network
Node size denotes gene connectivity while edge thickness stands for co-expression similarity.

Setting static=FALSE launches the interactive network. There is an interactive color bar to denote gene connectivity. The color ingredients must only be separated by comma, e.g. purple,yellow,blue, which means gene connectivity increases from purple to yellow. If too many edges (e.g.: > 300) are displayed, the network could get stuck. So the ‘Input an adjacency threshold to display the adjacency network.’ option sets a threthold to filter out weak edges. If not too many edges retained (e.g.: < 300), users can check ‘Yes’ under ‘Display or not?’, then the network would be responsive smoothly. To maintain acceptable performance, users are advised to choose a stringent threshold (e.g. 0.9) initially, then decrease the value gradually. The interactive feature allows users to zoom in and out, or drag a gene around. All the gene IDs in the network module are listed in ‘Select by id’ in decreasing order according to gene connectivity. Same with static mode, the target gene ID is appended ’_selected’.

If gene annotation is available in rowData slot and provided to ann argument, the annotation is seen by mousing over a node. In this example, Target.Description in rowData is provided to ann.

network(geneID="HRE2", data=se.fil.arab, ann='Target.Description', adj.mod=adj.mod, static=FALSE)

6 Shiny App

In additon to generating SHMs and the corresponding gene context plots from R, spatialHeatmap includes a Shiny App that provides access to the same functionalities from an intuitive-to-use web browser interface. Apart from being very user-friendly, this App conveniently organizes the results of the entire visualization workflow in a single browser window with options to adjust the parameters of the individual components interactively. For instance, genes can be selected and replotted in the SHM simply by clicking the corresponding rows in the expression table included in the same window. This representation is very efficient in guiding the interpretation of the results in a visual and user-friendly manner. For testing purposes, the spatialHeatmap Shiny App also includes ready-to-use sample expression data and aSVG images along with embedded user instructions.

6.1 Local System

The Shiny App of spatialHeatmap can be launched from an R session with the following function call.

shiny_all()

[ThG-Comment: many menu items are not functional in the Shiny App. E.g.: Instructions and Acknowledgement return error messages.]

The dashboard panels of the Shiny App are organized as follows:

  1. Left Side Panel: menu for workflow execution and parameter selection
  2. Expression Matrix: scrollable expression matrix uploaded by user
  3. Spatial Heatmap: aSVG image provided by user
  4. Matrix Heatmap: hierarchical clustering results for genes with similar expression profiles as target gene(s)
  5. Network Graph: interactive network module generated for genes with similar expression proviles as target gene(s)

A screenshot of the Spatial Heatmap component within the Shiny App window is shown below (Figure 11).

Screenshot of `spatialHeatmap's` Shiny App.

Figure 11: Screenshot of spatialHeatmap's Shiny App

After launching, the Shiny App displays by default one of the included data sets.

The gene expression data and aSVG image files can be uploaded to the Shiny App as tabular text (e.g. in CSV or TSV format) and SVG files, respectively. To also allow users to upload gene expression data stored in SummarizedExperiment objects, one can export them from R to a tabular file with the filter_data function. In this function call the user sets the desired directory path under dir. Within this directory the tabular file will be written to local_mode_result/processed_data.txt in TSV format. The column names in the exported tabular file preserve the experimental design information from the colData slot by concatenating the corresponding sample and condition information separated by double underscores. An example of this format is shown in Table 1.

To interactively access gene- or transcript-level annotations in the plots and tabels of the Shiny App, such as viewing functional descriptions by moving the cursor over network nodes, the corresponding annotation column needs to be present in the rowData slot and its column name assigned to the ann argument. In the exported tabular file the extra annotation column is appended to the expression matrix.8 Check for correctness. For readability I made a lot of changes in this paragraph.

se.fil.arab <- filter_data(data=se.aggr.sh, ann="Target.Description", sam.factor='samples', con.factor='conditions', pOA=c(0.03, 6), CV=c(0.30, 100), dir='./')

6.2 Server Deployment

As most Shiny Apps, spatialHeatmap can be deployed as a centralized web service. A major advantage of a web server deployment is that the functionalities can be accessed remotely by anyone on the internet without the need to use R on their local system. For deployment one can use local web servers or cloud services such as AWS, GCP or shinysapps.io. An example web instance for testing spatialHeatmap online is available here.

7 Supplement

To generate SHMs with custom data, proper formatting of the numeric data and/or aSVG images is often required. This section provides additional details on this topic.

7.1 Format the Data

The accepted data classes include vector, data frame, or SummarizedExperiment (SE). A vector applies to several numeric values measured for a single item (e.g. gene), and data frame applies to more items assayed in several samples and/or several conditions (e.g. 2 samples under 2 conditions). By contrast, SE applies to experiments with many samples and many conditions. Formatting the data is essentially define samples and/or conditions.

Vector
In the case of vector, the numeric values are measured from different samples. If one or more conditions are provided, the samples and conditions should be connected by double undescore, i.e. in the form of ’sample__condition’. If no conditions are provided, all the samples are assumed to have same condition, which is the toy example.

Take 2 samples occipital lobe and parietal lobe from the toy example for instance and assume there are 2 conditions, condition1 and condition2. Select 5 random values, assign 4 of them to the 2 samples under the 2 conditions, and the last one to a not-mapped sample. Note the value names should be unique.

# Random numeric values.
vec <- sample(x=1:100, size=5)
# Give unique names to random values.
names(vec) <- c('occipital lobe__condition1', 'occipital lobe__condition2', 'parietal lobe__condition1', 'parietal lobe__condition2', 'notMapped')
vec
## occipital lobe__condition1 occipital lobe__condition2 
##                         74                          7 
##  parietal lobe__condition1  parietal lobe__condition2 
##                         21                         50 
##                  notMapped 
##                         66

Plot SHMs.

spatial_hm(svg.path=svg.hum, data=vec, ID='toy', ncol=1, legend.r=1.2, sub.title.size=14)

Data Frame
In the case of data frame, numeric values are measured from different samples. Similarly, if one or more conditions are provided, the column names should be in the form of ’sample__condition’. If no conditions are provided, all the samples are assumed to have same condition.

Take the same samples and conditions in the vector case as example.

Make a numeric data frame of 20 rows and 5 columns. Name columns with the value names (each is unique) from above vector and rows with 20 genes (gene1, gene2, …, gene20).

# Make a numeric data frame.
df.test <- data.frame(matrix(sample(x=1:1000, size=100), nrow=20))
# Name the columns.
colnames(df.test) <- names(vec)
# Name the rows.
rownames(df.test) <- paste0('gene', 1:20)
# A slice of the data frame.
df.test[1:3, ]
##       occipital lobe__condition1 occipital lobe__condition2
## gene1                        667                        303
## gene2                        669                        666
## gene3                        133                        179
##       parietal lobe__condition1 parietal lobe__condition2 notMapped
## gene1                       831                       383        82
## gene2                       241                       578       699
## gene3                       561                       296       434

In the downstream interactive network, if users want to have a gene annotation by mousing over a node, a column of gene annotation can be appended to the data frame. For example, the 20 genes are annotated as ann1, ann2, …, ann20.

df.test$ann <- paste0('ann', 1:20)
df.test[1:3, ]
##       occipital lobe__condition1 occipital lobe__condition2
## gene1                        667                        303
## gene2                        669                        666
## gene3                        133                        179
##       parietal lobe__condition1 parietal lobe__condition2 notMapped  ann
## gene1                       831                       383        82 ann1
## gene2                       241                       578       699 ann2
## gene3                       561                       296       434 ann3

The pre-processing steps are optional and thus are skipped. Next plot SHMs on gene1.

spatial_hm(svg.path=svg.hum, data=df.test, ID=c('gene1'), ncol=1, legend.r=1.2, sub.title.size=14)

SummarizedExperiment

In the following, the same samples and conditions in the above data frame are taken as example.

Formatting data of SummarizedExperiment (SE, Morgan et al. (2018)) is essentially to make a targets file (a data frame of column metadata). The targets file usually has at least 2 columns that specifies sample and condition replicates respectively, and should be added to the colData slot. The data matrix should have assayed items (e.g. genes) and sample/conditions in rows and columns respectively, and must be in the assay slot. The rowData slot can store a data frame of annotaions corresponding to rows in assay slot, but is not required.

To plot spaital heatmap successfully, the targets file should meet the following requirements.

  1. It is a data frame and usually has at least one column of samples and one column of conditions. The rows correspond with columns in assay slot. If the condition column is not defined, the samples are assumped under same condition.

  2. The sample column specifies sample replicates. It is crucial that replicate names of the same sample must be identical. Otherwise, they are treated as different samples. E.g. occipital lobe, occipital lobe are the same sample while occipital lobe1, occipital lobe2 are different samples.

  3. The sample identifiers of interest must be identical with features of interest in aSVG respectively. It means even a dot, undescore, space, etc can make a difference and lead to target features not colored in SHMs. Since double underscore (__) is a reserved separator in spatialHeatmap, it cannot be used in sample or condition identifiers.

  4. The condition column has the same requirement with the sample column. E.g. condition1, condition1 is same conditoin while condition1A, condition1B is treated as different conditions.

In the following example, occipital lobe has 2 conditions condition1 and condition2, and each condition has 2 replicates, so there are 4 assays for occipital lobe. The same applies to parietal lobe. Based on this experiment design, the corresponding targets file is made, where a row is an assay.

# Two samples.
sample <- c(rep('occipital lobe', 4), rep('parietal lobe', 4))
# Two conditions.
condition <- rep(c('condition1', 'condition1', 'condition2', 'condition2'), 2)
# Targets file.
target.test <- data.frame(sample=sample, condition=condition, row.names=paste0('assay', 1:8))
target.test
##                sample  condition
## assay1 occipital lobe condition1
## assay2 occipital lobe condition1
## assay3 occipital lobe condition2
## assay4 occipital lobe condition2
## assay5  parietal lobe condition1
## assay6  parietal lobe condition1
## assay7  parietal lobe condition2
## assay8  parietal lobe condition2

Make a random numeric data frame of 8 columns and 20 rows. Each column is an assay and each row is a gene's expression profile. Columns must correspond with rows in targets file, so column names are assigned assay1-8.

# Make a numeric data frame.
df.se <- data.frame(matrix(sample(x=1:1000, size=160), nrow=20))
# Name the rows.
rownames(df.se) <- paste0('gene', 1:20)
# Replace the default column names. 
colnames(df.se) <- row.names(target.test)
# A slice of the data frame.
df.se[1:3, ]
##       assay1 assay2 assay3 assay4 assay5 assay6 assay7 assay8
## gene1    690    617    501    115    295    731    261    129
## gene2    952    146    569    497     68     16    546    564
## gene3    412    988    241    756    990    711     87    794
se <- SummarizedExperiment(assays=df.se, colData=target.test)
se
## class: SummarizedExperiment 
## dim: 20 8 
## metadata(0):
## assays(1): ''
## rownames(20): gene1 gene2 ... gene19 gene20
## rowData names(0):
## colnames(8): assay1 assay2 ... assay7 assay8
## colData names(2): sample condition

Similarly, in the downstream interactive network, if users want to have a gene annotation by mousing over a node, a data frame of gene annotation can be added to rowData slot, i.e. the ann column in df.test.

rowData(se) <- df.test['ann']

In this simple example, the normalization and filtering process is left out, but replicates should be aggregated. In function aggr_rep, the sample and condition columns in targets file are concatenated with double underscore to form ’sample__condition’ replicates for aggregating.

se.aggr <- aggr_rep(data=se, sam.factor='sample', con.factor='condition', aggr='mean')
## Syntactically valid column names are made!
assay(se.aggr)[1:3, ]
##       occipital.lobe__condition1 occipital.lobe__condition2
## gene1                      653.5                      308.0
## gene2                      549.0                      533.0
## gene3                      700.0                      498.5
##       parietal.lobe__condition1 parietal.lobe__condition2
## gene1                     513.0                     195.0
## gene2                      42.0                     555.0
## gene3                     850.5                     440.5

Plot SHMs on gene1.

spatial_hm(svg.path=svg.hum, data=se.aggr, ID=c('gene1'), ncol=1, legend.r=1.2, sub.title.size=14)

7.2 aSVG repository

The aSVG repository is from EBI Gene Expression Group, where the requirements on aSVG format are included. It contains aSVGs across different species and can be downloaded with funtion return_feature directly. If users cannot find a target aSVG in this repository, there is a step-by-step SVG tutorial to create custom aSVG images, which is developed by this project.

7.3 Update aSVG features

To change existing feature identifiers in aSVG, the function update_feature should be used. For testing purpose, an empty folder ~/test1 is created and a copy of the aSVG homo_sapiens.brain.svg packaged in spatialHeatmap is saved in there.

# Make an empty directory.
if (!dir.exists('~/test1')) dir.create('~/test1')
# Copy the "homo_sapiens.brain.svg" aSVG.
svg.hum <- system.file("extdata/shinyApp/example", 'homo_sapiens.brain.svg', package="spatialHeatmap")
file.copy(from=svg.hum, to='~/test1', overwrite=FALSE)

Use feature and species keywords to query the aSVG and return existing features, which is a data frame.

feature.df <- return_feature(feature=c('frontal cortex'), species=c('homo sapiens', 'brain'), dir='~/test1', remote=FALSE, keywords.any=FALSE)
feature.df

Make a vector of new feature identifiers corresponding to every returned feature, e.g. replacing spaces with dots. This vector must be added to the first column of the feature data frame, since that is where update_feature looks for new features. Then features are updated with update_feature.

# A vector of new features.
f.new <- c('frontal.cortex', 'prefrontal.cortex')

# New features added to the first column of feature data frame.
feature.df.new <- cbind(featureNew=f.new, feature.df)
feature.df.new

# Update the features.
update_feature(feature=feature.df.new, dir='~/test1')


# Version Informaion
sessionInfo()
## R version 4.0.0 (2020-04-24)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 16.04.5 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/libblas/libblas.so.3.6.0
## LAPACK: /usr/lib/lapack/liblapack.so.3.6.0
## 
## locale:
##  [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    
##  [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   
##  [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] stats4    parallel  stats     graphics  grDevices utils     datasets 
## [8] methods   base     
## 
## other attached packages:
##  [1] spatialHeatmap_0.99.0       ggplot2_3.3.0              
##  [3] GEOquery_2.56.0             ExpressionAtlas_1.16.0     
##  [5] xml2_1.3.2                  limma_3.44.1               
##  [7] SummarizedExperiment_1.18.1 DelayedArray_0.14.0        
##  [9] matrixStats_0.56.0          Biobase_2.48.0             
## [11] GenomicRanges_1.40.0        GenomeInfoDb_1.24.0        
## [13] IRanges_2.22.1              S4Vectors_0.26.1           
## [15] BiocGenerics_0.34.0         knitr_1.28                 
## [17] BiocStyle_2.16.0            nvimcom_0.9-25             
## 
## loaded via a namespace (and not attached):
##   [1] backports_1.1.7        rols_2.16.1            Hmisc_4.4-0           
##   [4] igraph_1.2.5           lazyeval_0.2.2         shinydashboard_0.7.1  
##   [7] splines_4.0.0          BiocParallel_1.22.0    digest_0.6.25         
##  [10] foreach_1.5.0          htmltools_0.4.0        magick_2.3            
##  [13] GO.db_3.11.1           gdata_2.18.0           magrittr_1.5          
##  [16] checkmate_2.0.0        memoise_1.1.0          cluster_2.1.0         
##  [19] doParallel_1.0.15      fastcluster_1.1.25     readr_1.3.1           
##  [22] annotate_1.66.0        prettyunits_1.1.1      jpeg_0.1-8.1          
##  [25] colorspace_1.4-1       blob_1.2.1             xfun_0.13             
##  [28] dplyr_0.8.5            crayon_1.3.4           RCurl_1.98-1.2        
##  [31] jsonlite_1.6.1         genefilter_1.70.0      impute_1.62.0         
##  [34] survival_3.1-12        iterators_1.0.12       glue_1.4.1            
##  [37] gtable_0.3.0           zlibbioc_1.34.0        XVector_0.28.0        
##  [40] scales_1.1.1           DBI_1.1.0              edgeR_3.30.0          
##  [43] Rcpp_1.0.4.6           viridisLite_0.3.0      xtable_1.8-4          
##  [46] progress_1.2.2         htmlTable_1.13.3       gridGraphics_0.5-0    
##  [49] flashClust_1.01-2      foreign_0.8-79         bit_1.1-15.2          
##  [52] preprocessCore_1.50.0  Formula_1.2-3          rsvg_2.1              
##  [55] htmlwidgets_1.5.1      httr_1.4.1             gplots_3.0.3          
##  [58] RColorBrewer_1.1-2     acepack_1.4.1          ellipsis_0.3.1        
##  [61] farver_2.0.3           pkgconfig_2.0.3        XML_3.99-0.3          
##  [64] nnet_7.3-14            locfit_1.5-9.4         dynamicTreeCut_1.63-1 
##  [67] labeling_0.3           later_1.0.0            ggplotify_0.0.5       
##  [70] tidyselect_1.1.0       rlang_0.4.6            AnnotationDbi_1.50.0  
##  [73] visNetwork_2.0.9       munsell_0.5.0          tools_4.0.0           
##  [76] RSQLite_2.2.0          fastmap_1.0.1          evaluate_0.14         
##  [79] stringr_1.4.0          ggdendro_0.1-20        yaml_2.2.1            
##  [82] bit64_0.9-7            caTools_1.18.0         purrr_0.3.4           
##  [85] mime_0.9               compiler_4.0.0         rstudioapi_0.11       
##  [88] curl_4.3               plotly_4.9.2.1         png_0.1-7             
##  [91] tibble_3.0.1           geneplotter_1.66.0     stringi_1.4.6         
##  [94] highr_0.8              lattice_0.20-41        Matrix_1.2-18         
##  [97] vctrs_0.3.0            pillar_1.4.4           lifecycle_0.2.0       
## [100] BiocManager_1.30.10    data.table_1.12.8      bitops_1.0-6          
## [103] grImport_0.9-3         httpuv_1.5.2           R6_2.4.1              
## [106] latticeExtra_0.6-29    promises_1.1.0         bookdown_0.19         
## [109] KernSmooth_2.23-17     gridExtra_2.3          codetools_0.2-16      
## [112] MASS_7.3-51.6          gtools_3.8.2           assertthat_0.2.1      
## [115] DESeq2_1.28.1          withr_2.2.0            GenomeInfoDbData_1.2.3
## [118] hms_0.5.3              grid_4.0.0             rpart_4.1-15          
## [121] tidyr_1.0.3            rmarkdown_2.1          rvcheck_0.1.8         
## [124] shiny_1.4.0.2          WGCNA_1.69             base64enc_0.1-3

8 Funding

This project has been funded by NSF awards: PGRP-1546879, PGRP-1810468, PGRP-1936492.

References

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